Expression of a tumor-specific antigen by a recombinant vector virus and use thereof in preventitive or curative treatment of the corresponding tumor

ABSTRACT

The invention concerns a vector being vaccinia virus, which comprises a heterologous DNA sequence which codes at least for the essential region of a tumor specific protein called T antigen, cloned within a non essential region of the vaccinia virus, as well as regulatory element required for the expression of said DNA sequence in higher cells, the vector is particularly useful as a pharmaceutical composition having a preventive or creative activity against tumors especially tumors caused by a papillomavirus.

This is a continuation of application Ser. No. 08/248,463, filed on May24, 1994, which was abandoned upon the filing hereof which is acontinuation of application Ser. No. 08/126,021, filed on Sep. 24, 1993,which is a continuation of application Ser. No. 07/984,242, filed Dec.1, 1992 which is a continuation of application Ser. No. 07/546,318,filed Jul. 2, 1990 which is a continuation in part of application Ser.No. 7/084,852, filed Aug. 13, 1987 all abandoned.

The present invention relates to new recombinant vaccinia viruses andtheir use as preventive or curative agents useful against tumors,especially tumors induced by papillomaviruses and more especiallyagainst human papillomaviruses types HPV-16, HPV-18, HPV-31, HPV-33,HPV-35, HPV-39 and HPV-45.

Tumor cells, whether they are spontaneous or induced by viruses, mayhave new antigens on their surface (Hellstrom, K. E., Hellstrom, I. &Brown, J. P., Springer Semin. Immunopathol. 5, 127-146, 1982).Tumor-specific antigens (T antigens) have already been used fordiagnosing (Herlyn, M., Blaszczyk, M. & Koprowski, H., Contr. Oncol. 19,160-170, 1984) and visualizing (Begent, R. H. J. Biochim. Biophys. Acta780, 151-166, 1985) human carcinomas.

More recently, their use as targets in a specific antitumor therapy hasbeen envisaged. The administration of anti-T antibodies, either in thefree form or bound to toxins or to radioactive isotopes, has alreadygiven encouraging results in the treatment of some clinical cases(Miller, R. A., Maloney, D., Warnke, R., McDougall, R., Wood, G.,Kawakami, T., Dilley, J., Goris, M. I. & Levi, R., In: Hybrodomas inCancer Diagnosis and Treatment, Mitchell, M. S. & Oettgen, H. F. (eds),Raven Press, New York, 1982).

Moreover, it is also possible to attempt to stimulate host-specificimmune responses against T antigens following different approaches suchas inoculating killed cells expressing T antigen (Gross, L., Cancer Res.3, 326-333, 1943; Foley, E. J., Cancer Res. 13, 835-837, 1953; Prehn, R.T. & Main, J. M., J. Natl. Cancer Inst. 18, 769-778, 1957), inducinganti-idiotypic antibodies directed against the variable region of anti-Timmunoglobulins (Lee, V. K., Harriott, T. G., Kuchroo, V. K., Halliday,W. J., Hellstrom, I. & Hellstrom, K. E., Proc. Natl. Acad. Sci., USA 82,6286-6290, 1985; Herlyn, D., Ross, A. H. & Koprowski, H. Sciences 232,100-102, 1986) or injecting the T antigen itself (Teventhia, S. S.,Flyer, D. C. & Tijan, R., Virology 107, 13-23, 1980). These experimentsare limited by the quantity of antigen available and by the fact thatthe stimulation of a cell type immune response, which is particularlyimportant in antitumor immunity, requires the simultaneous provision ofthe antigen and the histocompatibility determinants of the host(Zinkernagel, R. M. & Doherty, P. C., Adv. Immunol. 27, 51-177, 1979).

One object of the invention is to provide means for inducing, in vivo,an antitumor immunity.

One further object of the invention is to provide for expression of a Tantigen or a significant portion of the latter by a recombinant vacciniavirus, with the view of inducing, in vivo, an antitumor immunity.

The present invention relates to the use of a vaccinia virus as theexpression vector for a DNA sequence coding for the essential region ofa tumor-specific protein called T antigen.

A tumor-specific protein means an antigen which is specific for aspontaneous tumor and is absent in normal adult tissues, or an antigencoded by an oncogenic virus, the causative agents of the said tumor.

Essential region of the said protein is meant to denote the portion ofthe protein sequence capable of inducing an antitumor immunity or ofinducing a mechanism capable of causing the said tumor to regress.

Examples have been performed with different T antigens, in order toillustrate the wide scope of the invention.

First of all, T antigens which appear on the surface of rat cellstransformed by polyoma virus (PY) (Sjorgren, H. O., Hellstrom, I. &Klein, I, Cancer Res. 21, 329-337, 1961; Habel, K. Proc. Soc. Exp. Biol.Med. 106, 722-725, 1961; Ito, Y., Brocklehurst, J. R. & Dulbecco, R.,Proc. Natl. Acad. Sci., USA 74, 4666-4670, 1977) have been expressed bya vaccinia virus and shown to be effective against tumors induced by thepolyoma virus.

The polyoma virus induces several types of tumors in rodents. Tumorinduction involves the integration of the viral DNA into the host genomeand the expression of the early genes of the virus (Basilico, C.,Pharmac. Ther. 26, 235-272, 1984; Griffin, B. E. & Dilworth, S. M. Adv.Cancer Res. 39, 183-268, 1983; Tooze, J. DNA Tumor Viruses, Cold SpringHarbor Press, N.Y., 1981). The inoculation of rodents with killed cellstransformed by PY enables an immunity to be induced against a testinoculation with tumor cells induced by PY (Sjogren et al.; Habel etal.). However, the demonstration of the presence of tumor-specifictransplantation antigens and their relationship with T antigens, thesynthesis of which is controlled by the early region of the PY viralgenome, has not yet been clearly established. This study is complicatedby the fact that the early region of the PY genome codes simultaneouslyfor 3 separate proteins (Tooze et al.) called "large-T:LT","middle-T:MT" and "small-T:ST", in accordance with their respectivemolecular weights; these 3 antigens have the same N-terminal sequenceand are recognized by the same polyclonal antibodies.

As will be described in the examples, the 3 T antigens have been clonedand expressed separately, in order to define accurately their roles andtheir respective potentials.

The use of vaccinia virus as cloning and expression vector for foreignantigens has already been described (Panicali, D. & Paoletti, E., Proc.Natl. Acad. Sci. USA 79, 4927-4931, 1982; Mackett, M., Smith, G. L. &Moss., B., Proc. Natl. Acad. Sci. USA 79, 7415-7419, 1982). Recombinantviruses expressing antigens of heterologous viruses or of parasites havebeen employed to immunize animals against the corresponding pathogen(see review in Smith, G. L., Mackett, M. & Moss, B. Biotechnol. Genet.Eng. Rev. 2, 383-407, 1984). The antigens expressed by recombinantvaccinia virus are presented in the appropriate manner on the surface ofinfected cells and they enable a cell-type immune response to be induced(Wiktor, T. J., MacFarlan, R. I., Reagan., K. J., Dietzschold, B.,Curtis, P. J., Wunner, W. H., Kieny, M. P., Lathe, R., Lecocq, J. P.,Mackett, M., Moss, B. & Koprowski, H., Proc. Natl. Acad. Sci. USA 81,7194-7198, 1984; Wiktor, T. J., Kieny, M. P. & Lathe, R., Appl. Virol.2, in press, 1986; Bennik, J. R., Yewdell, J. W., Smith, G. L., Moller,C. & Moss, B., Nature 311, 578-579, 1984; McMichael, A. J., Michie, C.A., Gotch, F. M., Smith, G. L. & Moss, B., J. Gen. Virol. 67, 719-726,1986), which is particularly advantageous because it is known that theremoval of tumor cells involves cellular immunity (Hellstrom, K. E. &Hellstrom, I., Adv. Cancer Res. 12, 167-223, 1969; Heberman, R. B. Adv.Cancer Res. 19, 207-263, 1974).

The virus will comprise the whole range of elements required for theexpression of T protein in higher cells; it will comprise, inparticular, a promoter originating from the vaccinia virus. For example,the promoter of the 7.5 Kd protein gene can be employed. The promoter/Tcoding sequence assembly will be inserted into a non essential gene ofthe virus: for example, the gene for thymidine kinase (TK), which willenable the TK⁻ recombinant viruses to be selected easily.

The following examples illustrate the concept on which the invention isbased, in an animal model consisting of rat tumor cells and a T antigencoded by the polyoma virus PY which is responsible for tumor production;other examples are related to the expression of early non structuralgenes of papillomaviruses such as BPV and HPV. However, it is obviousthat similar results could be obtained with any other tumor-specificantigen which is absent or very poorly expressed in normal adult tissuesor which is encoded by an oncogenic virus, and that the use of thesedifferent T antigens also forms part of the invention.

Among other T antigens which may be employed as the target in theprocess according to the present invention, there may be mentionedantigens specific for colorectal carcinoma, melanoma, cancer of thekidney, neuroblastoma, carcinoma of the bladder, carcinoma of thebreast, lymphomas and adenomas of endocrine glands.

The tumor antigens which are used as target in the present invention maybe antigens coded by viruses responsible for the transformation of cellsinto cancer cells, in particular viruses such as papillomas or polyomas,or, more generally, constituents of the host, the expression of which isaltered in the tumor tissue.

Therefore, a further object of the invention is to produce activevaccines for preventive or curative purposes against tumors resultingfrom a pre-established infection by human papillomavirus (HPV).

Papillomaviruses represent a group of DNA viruses. They possess aprotein shell and a circular DNA genome of approximately 7900 basepairs. A number of types of papillomaviruses, bovine (BPV) and human(HPV), have been identified, and the genomes of some of these have beenfully sequenced (Pfister, H., 1987, in: The Papovaridiae: Thepapillomaviruses (editors, Salzman, N. P. and Howley, P. M.) PlenumPress, New York, p. 1-38).

Fundamental research work which has been carried out on these viruseshas thus led to an early region and a late region being distinguished intheir genome, by analogy with the polyoma and SV40 virus genome. Thelate region contains two reading frames L1 and L2, which code for themajor components of the capsid. The early region contains at least thereading frames E1, E2, E4, E5, E6, E7 and E8. The proteins encoded bythese reading frames possess different functions. Three of theseproteins are involved in the processes of oncogenic transformation ofinfected cells. The E5 protein of BPV-1, which possesses considerabletransforming power and which can transform cells in vitro independently(Schlegel, R. et al., 1986, Science 233, 464-467), is encoded by the 3'portion of the early region. The E6 protein of BPV-1 and E7 protein ofHPV-16 are encoded by the 5' portion of the early region, and areinvolved in the induction and maintenance of oncogenic transformation.These proteins appear to be derived from a common ancestral gene bysuccessive duplications of a peptide of 33 amino acids (Danos, O. andYaniv, M., 1987, in: Cancer Cells, 5: Papillomaviruses, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). The transforming power ofE7 has been demonstred for HPV-16 and 18 (Kanda, T. et al., 1988, J.Virol., 62, 610-613; Vousden, K. H. et al., 1988, Oncogene Res., 3, 1-9;Bedell, M. A. et al., 1987, J. Virol., 61, 3635-3640). Among the otherearly proteins, E1 and E2 possess a role in the replication and/orexpression of the virus, whereas no function has been demonstrated in E4and E8.

In man, HPVs are associated with pathological conditions ranging frombenign infection of the skin to warts and malignant tumors. Theseviruses are highly specific for the target tissues, in particular theepithelia of the epidermis of the genital, oral and respiratory tracts(Zur Hausen, H. and Schneider, A., 1987, in: The Papovaviridae: Thepapillomaviruses (editors, Salzman, N. P. and Howley, P. M.) Plenumpress, New York, p. 245-263). The epidemiological data strongly suggestthe role of certain strains of HPV in cancer of the neck of the uterusand of the lower passages (most frequently fatal tumor in women). HPV-16and HPV-18 DNA are found in most biopsies originating from genitalcancer cells; more rarely, HPV-31, HPV-33, HPV-35, HPV-39 and HPV-45 aredetected.

Pathological conditions associated with HPV viruses give rise to atherapeutic problem on account of their persistent and recurrent nature.Many approaches have already been used in the treatment of thesediseases: surgery, chemotherapy, antiviral agents and immunotherapy(Weck, P. K. and Whisnant, J. K., 1987, in: The papillomaviruses(editors, Sulzman, N. P. and Howley, P. M., Plenum Press, New York, p.393-402).

In particular, European patent publication EP-A-0 133 123 describes avaccination approach against an infection by papillomaviruses, whichconsists in preparing by genetic engineering proteins of the viralcapsid, that is to say structural proteins hence corresponding to thelate region of the virus, and in using them as immunogenic agents. Inthis document, the means described are directed towards protectionagainst an infection by the viruses themselves, and hence theoreticallyagainst all forms of infections liable to develop.

The present invention is directed more precisely to the production ofactive vaccines, for preventive or curative purposes against malignanttumors resulting from a pre-established infection by HPV.

The present invention is based on the observation that the formation oftumors induced by papillomaviruses is due to the expression of the earlygenes of the viruses.

The subject of the invention is hence a vaccinia virus which comprises aheterologous DNA sequence coding at least for the essential region of anon structural protein of a papillomavirus, as well as the regulatoryelements required for its expression in higher cells.

In view of the observations which have been made, and stated above,regarding the frequency of infection by type 16, 18, 31, 33, 35, 39 and45 HPV in cases of cancer of the neck of the uterus, the invention aimsmore especially at providing vaccinal compositions which are usefulagainst HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39 and HPV-45.

Thus, the subject of the invention is a vaccinia virus which comprisesat least one DNA sequence coding for at least one non-structural proteinof type 16, 18, 31, 33, 35, 39 or 45 HPV virus, and especially of theHPV-16 virus.

As stated above, the structure of the viruses of the papilloma family,especially BPV-1 and HPV, is such that there are at least 7 readingframes capable of corresponding to protein functions very specific tothe mechanisms of action of the virus and of maintenance of the viralgenome. Thus, it can be advantageous to prepare vaccinia viruses whichexpress several proteins simultaneously in the human body. This may beobtained either with several vaccinia viruses each expressing a givenprotein, or with one vaccinia virus containing several heterologous DNAsequences corresponding to the proteins chosen.

Naturally, the recombinant vaccinia virus will contain the assembly ofelements needed for expression of the proteins in higher cells, i.e.generally a transcription promoter of the gene and a region fortranslation initiation in the host cell; preferably, the promoter willusually be a promoter of a gene of the vaccinia virus used, such as thepromoter of the gene for the 7.5K protein of vaccinia virus, asindicated above. The promoter-coding sequence assembly will be insertedinto a non essential region of the virus, for example the thymidinekinase (TK) gene, thereby permitting ready selection of the TK⁻recombinant viruses.

The present invention also relates to pharmaceutical compositions forpreventing or curing tumors, containing an an active agent at least oneof the recombinant vaccinia viruses of the invention.

The vaccinal compositions according to the invention may be used for avariety of purposes. They may be used preventively, to prevent theappearance of malignant tumors, either before any infection by the virusor alternatively after an infection which has caused benign disorders,so as to avoid the situation where other tissues are attacked and becometransformed to cancerous tissues. They may also potentially be usedcuratively, to cause an already established tumor to disappear, or tocomplement or as a substitute for an ablation. These uses can relate toboth man and animals, especially cattle for the prevention of infectionsby BPV viruses. The recombinant vaccinia virus may be used, whereappropriate, with a pharmaceutically acceptable vehicle for itsinoculation into man or animals.

In general, the live virus is inoculated into man or animals.Nevertheless, it is also possible to envisage injecting into man oranimals the killed recombinant virus, presenting the chosen proteins atits surface, or the proteins purified from cell cultures infected by therecombinant vaccinia viruses.

The pharmaceutical compositions according to the invention may beprepared according to methods known in the vaccine field, and the dosesapplicable may vary over a wide range. They will be dependent, inparticular, on the patient's state and on other parameters which will beevaluated by the practitioner.

The invention will be illustrated by the examples below, which describeresults of vaccination on animals which have been injected with cellstransformed by polyomavirus (PY), bovine papillomavirus (BPV-1), and byhuman papillomavirus (HPV-16). In particular, the cloning and sequencingof the DNAs corresponding to the reading frames for the E5, E6 and E7proteins of HPV-16, as well as the results of vaccination obtained withrecombinant viruses expressing these proteins, are described.

BRIEF DESCRIPTION OF THE DRAWINGS

The detailed description of the invention will be accompanied by FIGS. 1to 12 which show:

FIG. 1 shows the structure of the early region of PY DNA anddemonstrates the 3 genes LT, MT and ST which overlap and, in particular,the corresponding portion of the N-terminal region which is common tothem. The transcription initiation site, in PY, is indicated by TATA andthe translation initiation and termination signals are denoted by ATGand TGA respectively. The position of introns as well as that of thepolyadenylation sites are indicated.

FIG. 2 shows the construction of three recombinant vaccine viruses, eachexpressing one of the T antigens of the PY virus.

FIG. 3 shows the immunofluorescence of cells infected with VV.PY.LT (A),VV.PY.MT (B), VV.PY.ST (C) and of uninfected cells (D).

FIG. 4 shows the SDS-PAGE gel of the immunoprecipitates of the productsof the reading frames E1, E2, E5 and E6 of the early region of thebovine papillomavirus BPV-1 genome.

FIG. 5 shows the latency period of the development of tumors in animalsvaccinated with the recombinant vaccinia viruses expressing the productof the reading frames E1, E2, E5, E6 or E7 of bovine papillomavirusBPV-1 (VVbE1, VVbE2, VVbE5, VVbE6 and VVbE7), or a control recombinantvaccinia virus VV-0 (not expressing a protein of BPV-1), and tested withFR3T3 cells transformed by BPV-1 (FR3T3 BPV-1-6).

FIG. 6 shows the latency period of the development of tumors in animalsvaccinated with the recombinant vaccinia viruses expressing the productsof the reading frames E5, E6and E7 of bovine papillomavirus BPV-1(VVbE5, VVbE6, VVbE7) or a VVbE5-VVbE6 or VVbE5-VVbE7 combination, or acontrolled recombinant vaccinia virus VV-0 (not expressing a protein ofBPV-1), and tested with FR3T3 cells transformed by BPV-1(FR3T3-BPV-1-3).

FIG. 7 shows diagrammatically the structure of the bacteriophage M13E7/E6 (HPV-16).

FIG. 8 shows diagrammatically the structure of the bacteriophage M13 E5(HPV-16).

FIG. 9 shows the complementary DNA sequence of the reading frame E5(HPV-16).

FIG. 10 shows the SDS-PAGE gel of the immunoprecipitates of the productsof the reading frames E6and E7 of the early region of the humanpapillomavirus HPV-16 genome.

FIG. 11 shows the changes in size of the tumors in animals vaccinatedwith the recombinant vaccinia virus expressing the product of thereading frame E6of human papillomavirus HPV-16 (VVhE6) and tested withprimary rat cells transformed by HPV-16 and a ras oncogene.

FIG. 12 shows the changes in size of the tumors in animals vaccinatedwith the recombinant vaccinia virus expressing the product of thereading frame E7 of human papillomavirus HPV-16 (VVhE7) and tested withprimary rat cells transformed by HPV-16 and a ras oncogene.

EXAMPLE 1

Construction of 3 recombinant vaccine viruses, each expressing one ofthe T antigens of the PY virus.

The coding sequences for the 3 T antigens of PY virus have already beencloned after their introns were removed by excision in vitro (1-2), togive the plasmids pPY-LT1, pPy-MT1, pPY-ST1 (2). These coding sequenceswere recovered by digestion with BglI at one end and HindII for LT,EcoRI for MT and PvuII for ST at the other end. The BglI end was madecompatible with a BamHI site by use of a single-stranded syntheticadapter (3.):

5'-d.GATCTGG-3'

The 3 DNA segments thus treated were introduced into the pTG186-polyvector between the BamHI-SmaI, BamHI-EcoRI and BamHI-SmaI sitesrespectively (FIG. 2). The pTG186-poly vector results from the insertionof M13TG131 polylinker (41) which provides several restriction sites tothe pTG1H-TK-7.5K vector (4) downstream of the promoter for the 7.5Kgene of the vaccine virus, which itself is inserted into the TK gene ofthe vaccine. The 3 coding sequences for T antigens and their translationinitiation and termination signals respectively have been inserted intoa nonessential gene of the vaccine virus, after being placed downstreamof a promoter for the vaccine virus. Following a double recombination,in cells infected with the wild virus and transfected with therecombinant plasmid, as has already been described previously (4), thecoding sequence for T antigen finds itself integrated into the genome ofthe vaccine-virus. The recombinant viruses carrying the foreign gene areselected for their TK⁻ character by multiplication in TK⁻ 143 B cells,in the presence of 5-bromodeoxyuridine.

The recombinant viruses, having integrated the coding sequences for Tantigens, have been identified by southern blotting, and 3representative viruses were chosen: VV.PY.LT-H, VV.PY.MT-I andVV.PY.ST-J.

EXAMPLE 2

In vitro study of the properties of T antigens synthesized by VV-PYrecombinant viruses.

The 3 T antigens expressed by the recombinant vaccine viruses arerecognized by antibodies directed against the native PY T antigen.

The intracellular localization of antigens was studied using fluorescentantibodies: semiconfluent hamster (BHK21) cells were infected separatelywith the types of VV.PY.T recombinant viruses (0.1 pfu/cell; incubationovernight); the T antigens were revealed,; in cells fixed with acetone(80% concentration), by the sequential application of hyperimmune ratantiserum, anti-T (RAF no. 4, CNRS-Nice (5); 1/50 PBS+1% bovineserumalbumin), followed by fluorescent antirat antibodies (goatimmunoglobulins, supplied by Miles, 1/100 PBS+1% bovine serum albumin).The 2 antibodies are each adsorbed for 20 minutes at 37° C. and thecells are then washed in order to remove the unbound antibodies. FIG. 3shows the immunofluorescence of cells infected with VV.PY.LT (A),VV.PY.MT (B), VV.PY.ST (C) and of uninfected cells (D).

It is seen from FIG. 3 a that, as expected (6), the LT protein islocated exclusively in the nucleus; the ST protein is located mainly inthe cytoplasm (FIG. 3c); the MT protein is located mainly in thecytoplasm (FIG. 3b) and not on the surface, as has been reported (7).Its detection, which is weak, but reproducible, in the perinuclearregion suggests an association with the Golgi apparatus and otherintracellular membranes, as has been indicated by other recent studies(8,9).

EXAMPLE 3

Study of the immune response induced in animals inoculatedsubcutaneously with the recombinant viruses.

In order to assess the vaccination potential of the 3 VV-PYrecombinants, their capacity to induce an antitumor immune response wasdetermined in vivo.

It is known that rats inoculated subcutaneously with syngeneic cellstransformed by PY quickly develop tumors located at the transplantationsite. This experimental model must enable the induction of an immuneresponse capable of blocking the development of the tumor to bedemonstrated.

Groups of 4-week old female rats (Fischer) were inoculatedsubcutaneously with the different recombinant viruses (at a dose of 10⁷pfu in 100 μl), reinoculated with the same dose after 12 days and thensubjected to a test inoculation, on the 16th day, with 3T3 rat cellstransformed by complete PY (PYT-21), at a dose of 2×10⁴ cells in 100 μl.One group of rats was inoculated with the 3 recombinant virusessimultaneously in order to demonstrate any cumulative effect of the 3 Tantigens, as has already been suggested (2).

The results are given in Table I:

All the nonvaccinated animals monitored develop tumors which can bedetected within 14 days from inoculating the transformed cells;additionally, no spontaneous regression is observed during theexperimental period (42 days).

The animals vaccinated with VV.PY.LT and VV.PY.MT develop small tumors(of a diameter >5 mm) which regress rapidly, to be totally eliminated in50 to 60% of animals.

The animals vaccinated with VV.PY.ST develop tumors which do not regresswith time. Therefore, the effect observed with VV.PY.LT and MT is quitespecific and cannot be attributed to a nonspecific stimulation of theimmune system by the vaccine virus, which is a very good immunogen andcould have had a mode of action similar to that of BCG (10).

The simultaneous inoculation of the 3 recombinants does not give asignificant improvement.

                  TABLE I                                                         ______________________________________                                        Rejection of tumors by rats inoculated sub-                                   cutaneously with VV.PY.T recombinants                                                     Number of animals rejecting the                                               tumor/total number inoculated                                     Vaccine  Time after                                                                             18       21    27     35                                    virus    challenge:                                                                             days     days  days   days                                  ______________________________________                                                          0/4      0/4   0/4    0/4                                   VV.PY.ST          0/7      0/7   0/7    0/7                                   VV.PY.MT          0/10     1/10  3/10   6/10                                  VV.PY.LT          0/10     0/10  2/10   5/10                                  VV.PY.LT          2/4      2/4   1/4    1/4*                                  +VV.PY.MT                                                                     +VV.PY.ST                                                                     ______________________________________                                         Note: *1 animal did not develop any detectable tumor until the 35th day       after the challenge.                                                     

The animals were sacrificed after 42 days and their sera were analysed(Table II). All the vaccinated animals have high antibody titers againstthe vaccine virus and against the cells transformed by PY, irrespectiveof whether they were capable of rejecting tumors or not.

Therefore, it seems that the rejection of tumors cannot be attributed tocirculating antibodies, but requires the participation of another immunemechanism.

                  TABLE II                                                        ______________________________________                                        Antibody titers in vaccinated and challenged animals,                         sacrificed after 42 days                                                                  Antibody titers* against                                                  Diameter of              the cells                                    Vaccine the tumor the            transformed                                  virus   (in mm)   vaccine (mean) by PY   (mean)                               ______________________________________                                                15        1.8            13.7                                                 20        6.9      (7.9) 10.2    (12.3)                                       15        15.9           7.3                                                   5        6.7            18.0                                         VV.PY.ST                                                                              15        253            56.5                                                 10        220     (224)  26.1    (37.0)                                       15        218            31.6                                                 20        203            33.8                                         VV.PY.MT                                                                              --        227            35.9                                                 --        258     (243)  32.6    (30.7)                                       --        266            28.4                                                 10        222            25.9                                         VV.PY.LT                                                                              --        226            11.8                                                 14        226     (243)  21.2    (37.9)                                       --        227            30.9                                                 --        295            87.8                                         VV.PY.LT                                                                              --        180            16.0                                         +VV.PY.MT                                                                             15        300     (247)  23.4    (22.8)                               +VV.PY.ST                                                                              3        252            33.9                                                  8        255            17.9                                         ______________________________________                                         *Antibodies were determined on microtiter plates with purified vaccine        virus (10.sup.6 pfu in 100 μl) or PYT21 cell suspension (10.sup.5 cell     in 100 μl); bound antibody is detected by the sequential addition of       sheep antirat IgG, labelled with biotin (Amersham) and then streptavidine     peroxidase (Amersham) followed by the ragent ELAVIAR9 (PasteurDiagnostic)     The absorption at 492 nm is measured with a Titertecuniscan plate reader.     Antibody titer is given by the product of multiplication of the measured      optical density by the dilution of the antiserum employed. Each result is     the mean value of 3 dilutions (1/50, 1/250 and 1/1,250).                 

EXAMPLE 4

Rejection of tumors in animals vaccinated intradermally with therecombinant viruses.

Another route for inoculating the vaccine virus was tried.

Groups of 4-week old female rats (Fischer) were vaccinatedintradermally, by scarifying the base of the tail with a scalpel, withpurified virus (10 μl of a 2×10⁹ pfu/ml stock).

A second dose is inoculated after 15 days. The animals are subjected totest inoculation, by injecting 2×10⁴ PYT-21 cells in 100 μlsubcutaneously, 4 days later.

The results are given in Table III.

                  TABLE III                                                       ______________________________________                                        Rejection of tumors by rats inoculated intradermally                          with the VV.PY.T recombinants                                                              Number of animals rejecting the                                               tumor/total number inoculated                                    Vaccine   Time after                                                                             22         29    39                                        virus     challenge:                                                                             days       days  days                                      ______________________________________                                                           0/4        0/4   0/4                                       VV.PY.ST           0/4        0/4   0/4                                       VV.PY.MT           2/8        7/8   8/8                                       VV.PY.LT           0/8        0/8   5/8                                       ______________________________________                                    

EXAMPLE 5

Treatment of tumor bearing animals with the VV.PY.T. recombinantviruses.

Recombinant virus administration was attempted as curative treatment fortumors.

10 rats which were inoculated with cells transformed by PY and which haddeveloped a tumor were inoculated with the VV.PY.LT recombinant after 12to 16 days; the tumor was 2 to 3 mm in size at the time of the firstinoculation.

In all the animals, the tumor continued to grow until it reached adiameter of 15 mm. In 2 animals, the tumor then regressed significantly(observation made on the 35th day) and was then completely eliminated.

No similar result was observed either with VV.PY.MT or with VV.PY.ST,which indicates differences in immunogenicity between the 3 types of Tantigen.

Deposition of a representative strain of the invention

The vector plasmid in which any coding sequence for a T antigen may beintegrated so as to be later recombined in the vaccine virus wasdeposited at the Collection Nationale de Cultures des Microorganismes(National Collection of Microbial Cultures) under the no. I 458, on Jun.20, 1985.

This E. coli plasmid derived from pML2 comprises a replication origin inE. coli, the β-lactamase gene, the vaccine TK gene, interrupted by the7.5K promoter of the vaccine and a following coding sequence (human IL2sequence), which can be exchanged with the sequence coding for a Tantigen.

EXAMPLE 6

Inhibition of the development of tumors induced by bovine papillomavirus(BPV-1), by vaccination with recombinant vaccinia viruses expressing theearly proteins of BPV-1.

a) Construction of vaccinia viruses expressing the early proteins ofBPV-1

Plasmid pm69 (11) contains the early region of the BPV-1 genome insertedas a HindIII-BamHI fragment into the HindIII and BamHI sites of plasmidpML2 (11). Subfragments containing the reading frames E1, E2, E5, E6 andE7 are excised by digestion with restriction enzymes (see Table I) andintroduced into the bacteriophages M13TG130 or M13TG131 (12) and thensubjected to an oligonucleotide-directed localized mutagenesis beforebeing transferred into plasmid pTG186 poly (13). These steps aresummarized in Table IV.

Legend to Table IV:

a!: the underlined nucleotides specify the bases not matched to theparent sequence of BPV-1

b!: the translation initiation codon and the restriction site which areused in the cloning are underlined.

c!: it was not possible to introduce the EcoRI-BamHI fragment of plasmidpM69 containing the reading frame E5into the bacteriophage M13TG131 inthe desired orientation: clones are obtained in the reverse orientationin which two independent plasmid fragments are cloned; theoligonucleotide-directed mutagenesis hence corresponds to the otherstrand of the DNA.

k!: end produced by digestion with a restriction enzyme followed bytreatment with the Klenow fragment of E. coli DNA polymerase I beforecloning;

na: not applicable.

                                      TABLE I                                     __________________________________________________________________________    Cloning and mutagenesis of subfragments of the                                BPV-1 genome for their transfer into vaccinia viruses.                        __________________________________________________________________________     BPV-1              Mutagenesis     Cloning                                   •                                                                        reading                                                                             •                                                                        vector   •                                                                          sequence (5'-3')                                                                             •                                                                        fragment                                   frame •                                                                        site        of oligonucleo-                                                                              •                                                                        vector                                    •                                                                        fragment           tides used  a! •                                                                        site                                                       •                                                                          sequences at the                                                              translation inititiaton site  b!                          E1     M13TG130                                                                              na                  BamHI-SstI                                 NruI-AvrII                                                                           BamHI!k!-XbaI               pTG186poly                                                (TCGCGAGCGTCATGG)   BamHI-SstI                                 E2     M13TG131                                                                              GAGGAGGATCCTGAAGAGGA                                                                              BamHI                                      EcoRI-SpeI                                                                           EcoRI-XbaI                  pTG186poly                                                (GGATCCTGAAGAGGATGG)                                                                              BamHI                                      E5     M13TG131                                                                              AGATTTGCCATAGTCGACCAGTCA c!                                                                       SalI-BamHI                                 EcoRI-BamHI                                                                          EcoRI-BamHI                 pTG186poly                                                (GTCGACTATGG)       SalI-BamHI                                 E6     M13TG130                                                                              CAGACCCCGGATCCAACATGGACCT                                                                         BamHI-XmaIII k!                            HpaI-SmaI                                                                            SmaI                        pTG186poly                                                (GGATCCAACATGG)     BamHI-SmaI                                 E7     M13TG130                                                                              TGCTAGGACTCGAGCAAACATGGTTCA                                                                       XhoI-EcoRI                                 HpaI-SmaI                                                                            SmaI                        pTG186poly,                                               (CTCGAGCAAACATGG)   SalI-EcoRI                                 __________________________________________________________________________

Oligonucleotide-directed localized mutagenesis permits the introductionof a translation initiation consensus sequence at the initiation codonso as to provide for the correct expression after transfer into vacciniavirus. In each case, the synthetic oligonucleotide introduces a singlerestriction site immediately before the sequence determining thebeginning of translation.

In the case of the reading frame of E1, it is not necessary to carry outa localized mutagenesis due to the presence of a restriction site in theBPV-1 genome (NruI site in position -11 relative to the initiation ATG(lacuna) and of a quasi-consensus sequence of the translation initiationregion (GCGTCATGG): the nucleotide G in position -3 is almost aseffective as A in translation initiation.

Primary chick embryo cells are cultured at 37° C. in an MEM medium(Gibco) supplemented with 10% of fetal calf serum. They are subjectedsimultaneously to an infection with a temperature-sensitive vacciniavirus and to a transfection with expression/transfer vectors carryingthe inserted segments of BPV-1 and wild-type vaccinia virus DNA. Afterselection, vaccinia recombinants in which the expression of the insertedsequence is under the control of the 7.5K promoter of vaccinia virus areisolated according to standard techniques.

The recombinant vaccinia viruses (VV) expressing the early proteins ofBPV-1 E1, E2, E5, E6 and E7 are referred to as VVbE1, VVbE2, VVbE5,VVbE6 and VVbE7 respectively.

b) Expression of E1, E2, E5, E6 and E7 in cells infected with therecombinant viruses

BHK-21 cells on Dulbecco's modified Eagle's medium MEM.BME (GIBCO)supplemented with 10% of fetal calf serum (GIBCO) are infected with oneof the six recombinant viruses at a multiplicity of approximately 20plaque forming units (pfu) per cell.

After one hour at 37° C., fresh medium is added and the cells areincubated for two hours. The medium is withdrawn and the cells arewashed once. MEM.BME medium without methionine and/or cysteine andsupplemented with 5% of dialyzed fetal calf serum is then added.Labeling is performed with 0.5-1 mCi/ml of ³⁵ S!-L-methionine and/or ³⁵S!-L-cysteine (Amersham) for 3 hours at 37° C. The cells thus labeledare rinsed twice with 20 mM Tris.HCl pH 7.2, 150 mM NaCl (TS buffer)containing aprotinin (1 IU/ml; Biosys, France), collected by scrapingand rinsed by two centrifugations in STE buffer (20 mM Tris.HCl pH 7.2,150 mM NaCl, 1 mM Na₂ EDTA, 1% aprotinin). The cells are lysed in RIPAbuffer 50 mM Tris.HCl, pH 7.4, 150 mM NaCl, 1 mM Na₂ EDTA (sic), 1%Triton X-100, 1% Na deoxycholate, 0.1% SDS! or fractionated to determinethe subcellular localization of the proteins of BPV-1.

Immunoprecipitation and SDS-PAGE are performed according to theprocedure described by Davis (14). Rabbit polyclonal antisera raisedagainst the bacterial fusion proteins corresponding to the readingframes E1, E2, E5, E6 and E7 are obtained by the conventional methodknown to those skilled in the art; see, for example, references (15) and(16).

Cells infected with VVbE1 produce a 69-kD nuclear protein which isspecifically recognized by anti-E1 antiserum (FIG. 4, strip 1, strip 2corresponds to an immunoprecipitation with a non-immune serum). Cellsinfected with VVbE2 contain a high level of a 48-kD polypeptidedisplaying a cross-reactivity with E2 and corresponding to the majortransactivation protein encoded by the reading frame E2 of BPV-1 (FIG.4, strip 3 and control strip 4). This polypeptide is detected in all thecell subfractions examined, in agreement with what has been describedfor the E2 protein of cells transformed by BPV-1 (17). VVbE6 codes for a15.5-kD protein specifically precipitated by anti-E6 serum (FIG. 4,strip 6 and control strip 7) and localized to the extent of more than50% in the cytoplasmic fraction, as for the E6 protein of cellstransformed by BPV-1 (18). Cells infected with VVbE5produce a 7.5-kDpolypeptide associated with the cell membranes (FIG. 4, strip 7 andcontrol, strip 8), a localization characteristic of the E5protein ofBPV-1 (19). Immunoprecipitation experiments with anti-E7 antiserumdisplay no cross-reactivity. Nevertheless, analysis of the expressionvectors reveals the presence of the reading frame of E7 in a correctorientation, and we assume that E7 is indeed produced but that, for someunknown reason, it is not precipitated by the antiserum available.

c) Vaccination against tumor cells induced by BPV-1

Groups of 4-week-old female rats (Fischer) are inoculated intradermallyor intraperitoneally with the different recombinant viruses (at a doseof 10⁸ pfu in 100 μl). They undergo a booster injection with the samedose after 12 days, and are subjected to a challenge inoculation betweendays 16 and 17 with Fischer rat 3T3 syngenic line (FR3T3) cellstransformed by BPV-1 according to the protocol described in references(11, 20) and referred to as FR3T3-BPV-1-6. For this purpose, 2×10⁴ cellsare injected subcutaneously in a volume of 200 μl of MEM.BME mediumwithout serum.

The results are shown in FIG. 5:

all the animals monitored vaccinated with VV-0 develop tumors after alatency period of 43 days on average.

the animals vaccinated with VVbE1 or VVbE2 show no modification in thedevelopment of tumors.

the animals vaccinated with VVbE5, VVbE6 and VVbE7 show a significantdelay in the appearance of tumors. In addition, in a few cases, there isno development of tumors (>250 days after the challenge), points shownin the box at the top of the figure.

These experiments are repeated, performing the challenge inoculationwith FR3T3 line cells transformed by BPV-1 according to the protocoldescribed in references (11, 20) and referred to as FR3T3-BPV-1-3. SinceVVbE1 and VVbE2 are without effect, only VVbE5, E6 and E7 are tested,and two groups of rats are inoculated with 2 recombinant virusessimultaneously, either VVbE5and VVbE7, or VVbE5and VVbE6, in order todetect a possible cumulative effect of these antigens. FIG. 6 confirmsthe beneficial effect of a vaccination with VVbE5and VVbE7 (points inthe box at the top of the figure correspond to the animals which do notdevelop a tumor), whereas VVbE6 remains without a significant effect inthis situation. In addition, no pronounced cumulative effect is seenwith the VVbE5, VVbE7 combination.

EXAMPLE 7

Cloning of the HPV-16 virus genome.

CaSki line cells (ATCC 1550) contain the HPV-16 virus genome integratedin a chromosome. From 10 75-cm³ flasks of semi-confluent CaSki cells,the total genomic DNA is purified in the following manner: the cells arerecovered by scraping, centrifuged and washed, and then taken up in 15ml of TE buffer 10 mM Tris pH 7.5, 1 mM EDTA! +0.5% SDS. After treatmentwith proteinase K (7.5 mg/15 ml) at 37° C. for 16 hours, the DNA isstored at 4° C. In this manner, 1 ml of solution containing 0.6 mg ofDNA is obtained.

70 μg of this DNA are subjected to a partial digestion with the enzymeMboI (10 min; 40 units) and then deposited on a linear sucrose gradient(20%-40%). After centrifugation (rotor SW28, 16 hours at 25,000 rpm, 20°C.), 500-μl fractions are collected. The fractions containing DNA ofsize between 10 and 20 kb (fraction 23-26) are combined, dialyzedagainst TE buffer and then precipitated with ethanol. In this manner, 5μg of DNA are obtained.

In order to have a cloning vector for the DNA described above,bacteriophage lambda EMBL 301 DNA (21) is digested with the restrictionenzyme BamHI. After phenol/chloroform extraction and ethanolprecipitation, the DNA is resuspended in TE buffer.

The CaSki cell genomic DNA and bacteriophage lambda DNA are then ligatedaccording to a conventional protocol (1 μg of vector, 2 μg of genomicDNA). After packaging of the DNA by means of a kit marketed by Amersham,a total of 0.75×10⁶ independent clones is obtained. These bacteriophagesare plated with E. coli Q358 bacteria on 18 dishes 14 cm in diameter andon LBM medium in order to be screened with synthetic oligonucleotidesdeduced from the HVP-16 virus sequence (EMBL) PA16!.

The screening of the recombinant bacteriophage DNA is performed in aconventional manner for those skilled in the art. The oligonucleotides1817 (sequence 5' CATGCATGGAGATACACCTACATTG 3'; reading frame E7(lacuna) and 1818 (sequence 5' GTGGATAACAGCAGCCTCTGCGTTT 3'; readingframe E5), radioactively labeled with ³² P, are mixed and hybridizedwith the phage DNA (transferred onto nitrocellulose filters) for 16hours at 55° C. in a 6-fold concentrated SSC buffer. The filters arethen washed under the same stringency conditions; 10 positive signals(1-10) were obtained. The area of the Petri dishes corresponding tothese signals is taken up in 1 ml of LBM buffer. These suspensions arereplated on Petri dishes 10 cm in diameter (2-10 μl of suspension perdish) in duplicate and a secondary screening is performed with theoligonucleotides 1817 and 1818 separately. Since the signalscorresponding to the phages obtained from the first isolations 4 and 5are the most intense, these two clones are chosen for the subsequentexperiments.

Minicultures of E. coli Q358 bacteria, infected with the bacteriophageisolated from the subclones 4-1, 4-2, 5-1 and 5-2, are prepared. The DNAof these clones is purified and subjected to digestion with therestriction enzyme PstI in order to analyze the recombinants selected.

After migration on a 1% agarose gel, the above DNAs are transferred ontoa nitrocellulose membrane according to Southern's technique. Thesemembranes are then incubated with the two oligonucleotides 1817 and 1818labeled with ³² P by the action of polynucleotide kinase, as above.After hybridization and washing of the membrane, the latter is subjectedto autoradiography. Two bands corresponding to DNAs of sizes 1063 and1772 bp, respectively, are thereby visualized, indicating that theclones 4-1, 4-2, 5-1 and 5-2 possess the HPV-16 virus genome.

EXAMPLE 8

Sequencing of the DNAs corresponding to E6, E7 and E5

The bacteriophage 5-1 is chosen for the subsequent experiments. In orderto have large amounts of the DNA of this clone, a DNA preparation from500 ml of infected E. coli Q358 bacteria is made according to aconventional protocol. 800 μg of DNA are thereby obtained. Afterdigestion with the enzyme PstI, the DNA is subjected to electrophoresison 1% agarose gel containing ethidium bromide and visualized with a UVlamp. The gel bands corresponding to the sizes 1063 and 1772 bp are cutout and the DNA is extracted using a "Geneclean" kit (Bio101 Inc). ThisDNA is then ligated in a bacteriophage M13TG130 opened at the PstI site,and transformed into competent E. coli NM522 bacteria.

The recombinant bacteriophage M13 DNA is purified from minicultures (1.5ml) and analyzed by digestion with restriction enzymes. A clonecontaining the 1063 bp HPV-16 band (M13 E5) and a clone containing the1772 bp HPV-16 band (M13 E7/E6) are selected.

In order to limit the work of sequencing, and since the desired DNAs arethose corresponding to the reading frames E5, E6 and E7, only theregions of the PstI restriction fragments corresponding to the E6 and E7proteins (M13 E7/E6) and E5protein (M13 E5) are sequenced, by means ofsynthetic oligonucleotides of sequence

5' ATCTAACATATATTC 3' and 5' GTTGTTCCATACAAA 3' for M13 E7/E6

and

5' GTCTGCCTATTAATAC 3' for M13 E5.

FIG. 7 shows the structure of the bacteriophage M13 E7/E6 and FIG. 5that of the bacteriophage M13 E5. The sequence obtained for E7 reveals atotal homology with the sequence contained in the libraries of PPH16data. For E6, two mutations are observed: G in place of A in position+46 and G in place of T in position +264, relative to the initiation ATGof E6. For E5, the sequence shown in FIG. 9, which differs by a fewbases from the sequence contained in the library of data, is obtained.

EXAMPLE 9

Cloning of the DNA fragments carrying E6, E7 and E5into the transfervector.

Before integrating the DNAs coding for these 3 reading frames inrecombinant vaccinia viruses, it is necessary to modify them in order tohave single restriction sites upstream and downstream from the genes,and to improve the sequences around the initiation ATG in order toobtain a good translation and a high level of synthesized protein.

a) Reading frame E6

A SalI restriction site and a SphI restriction site are created upstreamand downstream, respectively, from the coding sequence for E6 by meansof two synthetic oligonucleotides, employing a technique ofoligonucleotide-directed localized mutagenesis (Amersham kit). These twopoint mutations are produced simultaneously using the followingoligonucleotides:

5' GTTAGTATAAAAGTCGACCCACCATGCACCAAAAGAG 3'

5' CATGCATGCAGATACACC 3'

A nucleotide A in position -3 relative to the ATG is introduced in placeof a nucleotide T at the same time as the SalI site.

The bacteriophages obtained are analyzed by digestion with restrictionenzymes and by sequencing. A clone is selected for the subsequentexperiments, designated M13TG1181. The SalI-SphI restriction fragment isthen cloned into the vector pTG186 poly (13) opened at the SalI and SphIsites (pTG2198). This vector enables the DNA to be transferred into thevaccinia virus genome.

b) Reading frame E7

A PstI restriction site and a nucleotide A in position -3 are introducedupstream from the sequence coding for the E7 protein by localizedmutagenesis by means of the following oligonucleotide:

5' GTAGAGAAACCCTGCAGCCACCATGCATGGAG 3'

Several clones were obtained. One of them, which contains a 350-bp PstIrestriction fragment, is sequenced in the region corresponding to themutation. The sequence indeed corresponds to the expected mutation(M13TG1182). The PstI restriction fragment is then inserted into plasmidpTG186 poly open at the PstI site (pTG2199).

c) Reading frame E5

A PstI restriction site is introduced upstream from the initiation ATGof the reading frame E5(position 3851 in PPH16) by localized mutagenesisby means of the following oligonucleotide:

5' GTCTACTGGATTTACTGCAGTATGACAAATCTTGAT 3'

An EcoRI restriction site is introduced downstream from the stop codonby means of the following oligonucleotide:

5' GTATATGTACATAATGAATTCTTACATATAATTGTTG 3'

These two mutations are introduced simultaneously (Amersham kit). Theclone selected for the subsequent experiments is designated M13TG 3151.The PstI-EcoRI restriction fragment is then inserted into plasmid pTG186poly opened at the PstI and EcoRI sites (pTG3180).

EXAMPLE 10

Construction of the recombinant vaccinia viruses.

Plasmids pTG2198, 2199 and 3180 are used for transferring the readingframes E6, E7 and E5into a vaccinia virus (Copenhagen strain) asdescribed above. The recombinant viruses in which the coding sequencesare under the control of the 7.5K promoter of vaccinia are referred toas VVhE6, VVhE7 and VVhE5.

BHK-21 cells on MEM.BME medium supplemented with 10% of fetal calf serumare infected with one of the three recombinant viruses at a multiplicityof approximately 20 pfu per cell.

After incubation according to the protocol described above, the cellsare recovered, lysed and fractionated in order to determine thesubcellular localization of the proteins of HPV-16.

The expression of the E6 and E7 genes is verified by immunoprecipitationof the proteins (labeled with ³⁵ S !cysteine) synthesized.

Polyclonal antibodies raised against the bacterial fusion proteinscorresponding to the reading frames of E6 and E7 are obtained by theconventional method known to those skilled in the art and described inreferences (22) and (23).

Cells infected with VVhE6 produce an 18-kD cytoplasmic protein which isspecifically recognized by anti-E6 antiserum (FIG. 10, strip 1, strip 2corresponds to an immunoprecipitation with a non-immune serum). Cellsinfected with VVhE7 also produce a 19-20-kDa cytoplasmic proteinspecifically recognized by anti-E7 antiserum (FIG. 10, strip 3, strip 4corresponds to an immunoprecipitation with a non-immune serum).

For E5, since no antiserum is available, the integration of theexpression block E5 into the vaccinia virus is verified by DNA analysisaccording to Southern's technique.

EXAMPLE 11

Effect of vaccination with VVhE6 and VVhE7 on the development of tumorsinduced by HPV-16.

Groups of 4-week-old female rats (Fischer) are vaccinated by intradermalinjection of 5×10⁷ pfu (in 100 μl) of the different recombinant viruses.They are subjected to a booster injection with the same dose after 12days.

Between days 16 and 17, they are challenged with a line obtained bycotransfection in primary rat cells: (RE01 line).

1) by a plasmid containing the HPV-16 genome under the control of theLTR of Moloney's retrovirus (24);

2) by a plasmid coding for a gene for resistance to G418 and for EJ-RAS,which transforms only established cell lines or lines expressing animmortalizing gene.

2×10⁴ cells are injected subcutaneously in a volume of 200 μl of MEM.BMEbuffer without serum.

From FIGS. 11 and 12, the following observations can be made:

the unvaccinated control animals develop tumors detectable 10 days afterthe inoculation of the transformed cells (see dashed lines).

the animals vaccinated with VVhE6 do not develop a tumor in 2 cases outof 7 (>100 days). When tumors appear, in 3 cases their development isslowed down very significantly, and for 2 animals the appearance of thetumors is delayed (24 and 34 days instead of 10 days). Finally, in twocases, the development of the tumors is identical to that shown by theanimals in the control batch.

the animals vaccinated with VVhE7 do not develop a tumor in 3 cases outof 7 (>100 days). In the other 4 cases, the tumors appear from day 10onwards, but their development is significantly retarded.

In order to test the protective effects of these vaccinations, theanimals which, in the two batches, did not develop tumors are takenagain and challenged 80 days after the first inoculation challenge with10 times the initial dose, that is to say 2×10⁵ cells. Under suchconditions of inoculation, control animals develop tumors after 4 days.

the animals which had been vaccinated with VVhE6 develop tumors in anidentical manner to the control animals.

in contrast, the animals previously vaccinated with VVhE7 do not developtumors (>100 days) even under these conditions.

These results, as well as those stated above, bring out the importantpart played by the E7 antigens in protection against the development oftumors induced by HPV-16.

In order to verify the above results, two other series of experimentsare carried out with other cell lines. Groups of 4-week-old female ratsare vaccinated by intradermal injection of the recombinant viruses, asdescribed above. The rats vaccinated with the recombinant viruses arechallenged with 2×10⁴ or 10⁵ primary rat cells (RE31 line) preparedaccording to the protocol described above. The number of animals whichrejected the tumors, or in which a delay is observed in the appearanceof the latter, is given in the first part of Table V (First challenge).In this table, VV0 corresponds to a control recombinant vaccinia virusnot expressing a protein of HPV). Thereafter, rats which have rejectedthe tumors a first time are again challenged, but using a higherconcentration of primary cells (2×10⁵ instead of 2×10⁴ or 10⁵). Theresults obtained are presented in the second part of Table V (Secondchallenge).

                  TABLE V                                                         ______________________________________                                                               Delay in the                                           Recombinant            appearance of                                          virus     Rejection    the tumor  Total                                       ______________________________________                                        First challenge by injection of 2 × 10.sup.4 RE31 line cells per        rat                                                                           VVhE6     3/18         5/18       8/18                                        VVhE7     3/17         5/17       9/17                                        VV0       0/17         0/17       0/17                                        First challenge by injection of 10.sup.5 RE31 line cells per rat              VVhE6     7/20         1/20       8/20                                        VVhE7     5/20         0/20       5/20                                        VV0       1/20         0/20       1/20                                        Second challenge by injection of 2 × 10.sup.5 RE31 line cells per       rat                                                                           VVhE6     2/4          0/4        2/4                                         VVhE7     3/4          1/4        4/4                                         VV0       0/5          0/5        0/5                                         ______________________________________                                    

In another series of experiments, the primary rat cell lines used forchallenging the vaccinated rats are cotransfected with:

1) a plasmid containing the HPV-16 genome under the control of its ownpromoter,

2) a plasmid coding for a gene for resistance to G418 and for EJ-RAS,which transforms only established cell lines or lines expressing animmortalizing gene. The line RE604 is thereby obtained. Table VIpresents the results obtained under these experimental conditions.

                  TABLE VI                                                        ______________________________________                                        Challenge by injection of 2 × 10.sup.4 RE604 line cells per rat                                Delay in the                                           Recombinant            appearance of                                          virus      Rejection   the tumor  Total                                       ______________________________________                                        VVhE6      0/30        6/30       6/30                                        VVhE7      7/40        10/40      17/40                                       VVhE6/VVhE7                                                                              0/10        2/10       2/10                                        VV0        0/30        0/30       0/30                                        ______________________________________                                    

The results of these experiments bring out the important part played bythe E7 and E6 antigens in protection against the development of tumorsinduced by HPV-16.

In all the cell lines, the expression of the E7 protein is verified byimmunoprecipitation. The protective action exerted by the E7 antigen ishence not dependent on the line under study.

The following strains were deposited on Feb. 24, 1989 at the CollectionNationale de Cultures de Microorganismes (National Collection ofMicroorganism Cultures) of the Pasteur Institute (Paris):

E. coli pTG2198 under No. I-837.

E. coli pTG2199 under No. I-838.

E. coli pTG3180 under No. I-839.

REFERENCES

1. Treisman, R. Novak, U., Favaloro, J. & Kamen, R. Nature 292, 595-600(1981).

2. Rassoulzadegan, M., Cowie, A., Carr, A., Glaichenhaus, N., Kamen, R.& Cuzin, F. Nature 300, 713-718 (1982).

3. Lathe, R., Balland, A., Kohli, V. & Lecocq, J. P. Gene 20, 187-195(1982).

4. Kieny, M. P., Lathe, R., Drillien, R., Sphener, D., Skory, S.,Schmitt, D., Wiktor, T. J., Koprowski, H. & Lecocq, J. P. Nature 213,163-166 (1984).

5. Clertant, P., Gaudray, P., May, E. & Cuzin, F. J. Biol. Chem. 259,15196-15203 (1984).

6. Takemoto, K. K., Malmgrem, R. A. & Habel, K. Virology 28, 485-488(1966).

7. Schaffhausen, B. S., Dorai, H., Arakere, G. & Benjamin, T. L. Molec.Cell. Biol. 2, 1187-1198 (1982).

8. Zhu, Z. Veldman, G. M., Cowie, A., Carr, A., Schaffhausen, B. &Kamen, R. J. Virol. 51, 170-180 (1984).

9. Dilworth, S. M., Hansson, H. A., Darnfors, C., Bjursell, G., Streuli,C. H. & Griffin, B. E. Embo, J. 5, 491-499 (1986).

10. Old, L. J., Clarke, D. A. & Benacerraf, B. Nature 184, 291-292(1959).

11. Binetruy, B. et al. (1982). Embo J. 82, 621-628.

12. Kieny, M. P. et al (1983). Gene 26, 91-99.

13. Kieny, M. P. et al (1984). Nature 312, 163-166.

14. Davis G. et al (1986). Bacis methods in molecular biology(Elsevier).

15. Androphy, E. J. et al (1987) in: The papovaviridae: Thepapillomaviruses (editors, Salzman, N. P. and Howley, P. M.) PlenumPress, New York, p. 79-85).

16. Schlegel, R. and Wade-Glass, M. (1987) in: The papillomarivuses(editors Salzman), N. P. and Howley, P. M.) Plenum Press, New York,p.87-91.

17. Schiller et al (1984). PNAS 81, 7880.

18. Androphy, E. J. et al. (1986). Science 230, 442-445.

19. Burckhardt, A. et al. (1987). Embo J. 6, 2381-2385.

20. Grisoni, M. et al (1984). Virology 135, 406-416.

21. Lathe, R., et al (1987). Gene 57, 193-201.

22. Androphy, E. J. et al (1987). Embo J. 6, 989-992.

23. Shotkin and Wettstein (1986), PNAS 83, 1689-1694.

24. Storey, A. et al (1988), Embo J. 7, 1815-1820.

We claim:
 1. A method of treating tumors using recombinant vacciniaviruses encoding an immunogenic part of a tumor-specific antigen of avirus that induced said tumors, comprising the steps of:a) preparing arecombinant vaccinia virus comprisingi) a heterologous DNA sequencewhich codes at least for a region essential for inducing an immuneresponse to the tumor-specific antigen and ii) regulatory elementsrequired for expression of said DNA sequence in higher cells, whereinsaid heterologous DNA sequence and said regulatory elements are clonedwithin a non-essential region of said vaccinia virus; and b)administering said recombinant vaccinia virus to humans or animalshaving said tumors.
 2. The method of claim 1, wherein said recombinantvaccinia is administered to humans or animals as a pharmaceuticalproduct having a preventive activity against tumors or anti-tumoractivity.
 3. The method of claim 2, wherein said pharmaceutical productcontains a pharmaceutically acceptable vehicle enabling it to beadministered by injection into humans or animals.
 4. The method of claim1, wherein said DNA sequence codes for a protein encoded by an oncogenicvirus.
 5. The method of claim 4, wherein said DNA sequence originatesfrom an oncogenic DNA virus or is a DNA copy from an oncogenic DNAvirus.
 6. The method of claim 5, wherein said DNA sequence originatesfrom a virus selected from the group consisting of papovaviruses andretroviruses.
 7. A method of claim 6, wherein said DNA sequenceoriginates from a papovavirus selected from the group consisting ofpapillomavirus and polyomaviruses.
 8. A method of treating tumors usingrecombinant vaccinia viruses encoding an immunogenic part of atumor-specific antigen of a virus that induced said tumors, comprisingthe steps of:a) preparing a recombinant vaccinia virus comprisingi) aheterologous DNA sequence which codes for at least an essential regionof a non-structural protein from a papillomavirus and ii) regulatoryelements required for expression of said DNA sequence in higher cells,wherein said heterologous DNA sequence and said regulatory elements arecloned within a non-essential region of said vaccinia virus; and b)administering said recombinant vaccinia virus to humans or animalshaving said tumors.
 9. The method of claim 8, wherein said regulatoryelements comprise a transcription promoter and translation initiationand termination signals.
 10. The method of claim 9, wherein saidtranscription promoter originates from said vaccinia virus.
 11. Themethod of claim 10, wherein said promoter is the promoter of the 7.5Kprotein gene of the vaccinia virus.
 12. The method of claim 9, whereinsaid DNA sequence comprises the translation initiation and terminationsignals of vaccinia virus.
 13. The method of claim 12, wherein saidtranslation initiation signal contains an A or a G in position-3. 14.The method of any of claims 8 to 13, wherein said DNA sequence codes forat least one early protein of the early proteins of an HPV-16 virus. 15.The method of claim 14, wherein said early proteins are selected fromthe group consisting of E1, E2, E4, E5, E6, and E7.
 16. The method ofclaim 15, wherein said early proteins are selected from the groupconsisting of E6, E7, and E5.
 17. The method of claim 16, wherein saidearly protein is the E7 protein.
 18. The method of claim 16, whereinsaid early protein is the E5protein whose coding sequence is shown inFIG.
 9. 19. The method of claim 16, wherein said early protein is the E6protein.
 20. The method of claim 14, wherein said DNA sequence is clonedwithin a thymidine kinase (TK) gene.
 21. The method of any of claims 1,4-6, 8-10 or 15-18, wherein said DNA sequence is cloned within athymidine kinase (TK) gene.
 22. A method of treating tumors usingrecombinant vaccinia viruses encoding an immunogenic part of a tumorspecific antigen of a virus that induced said tumors, comprising thesteps of:a) preparing a recombinant vaccinia virus comprising:i) aheterologous DNA sequence which codes for at least an essential regionof a T antigen protein from a polyomavirus, and ii) regulatory elementsrequired for expression of said DNA sequence in higher cells, whereinsaid heterologous DNA sequence and said regulatory elements are clonedwithin a non-essential region of vaccinia virus; and b) administeringsaid recombinant vaccinia virus to humans or animals having said tumors.23. The method of claim 22, wherein said regulatory elements comprise atranscription promoter and translation initiation and terminationsignals.
 24. The method of claim 23, wherein said transcription promoteroriginates from said vaccinia virus.
 25. The method of claim 24 whereinsaid promoter is the promoter of the 7.5K protein gene of the vacciniavirus.
 26. The method of any one of claims 12, 23, 24 or 25, whereinsaid DNA sequence is cloned within a TK gene.